Our long term goal is to develop and exploit new strategies for the design and synthesis of biological catalysts with tailored specificities. The specific aim of this proposal is to design, synthesize, and characterize sequence-specific hybrid ribonucleases and deoxyribonucleases with defined binding site sequences and binding site sizes. A new binding site consisting of an oligodeoxyribonucleotide of defined length and sequence has been site-selectively introduced outside the catalytic sites of the relatively nonspecific enzymes, bovine pancreatic ribonuclease A and staphylococcal nuclease. The resulting hybrid enzymes are able to sequence-specifically cleave single-stranded DNA and RNA. The design of this first generation of hybrid enzymes will be optimized and the catalytic properties and specificities characterized in detail. In addition, strategies will be explored for cleaving double- stranded DNA with these and other hybrid enzymes,including rec A mediated triple-strand formation, triple-helix formation, and derivatization of DNA binding proteins with staphylococcal nuclease. These hybrid sequence-specific phosphodiesterases will provide important tools for studies of both RNA and DNA structure and function as well as for mapping, isolating, and cloning nucleic acids. The strategies developed here may be applied to the design of other hybrid enzymes with tailored specificities for application in chemistry, biology, and medicine.